Human neutralizing antibody pair blocks COVID-19-ACE2 binding

By Dr. Liji Thomas, MDMay 10 2020

With the ongoing COVID-19 pandemic carving its deadly path across the world, and no end in sight in the immediate future, researchers are working furiously to bring out an effective therapeutic drug or vaccine that will stop the virus in its tracks.

A new study published on the preprint server medRxiv* in May 2020 reports that a two-antibody combination is capable of neutralizing the virus. This could open the way for the development of a new therapy.

The S Protein

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is more easily transmitted from person to person than earlier zoonotic coronaviruses, perhaps because of the differences in its genotype. There are five human pathogens from the betacoronavirus family, including the SARS and MERS viruses, which caused severe outbreaks of disease over the past few decades.

The spike (S) glycoprotein on the surface of the virus exists as a trimer, which is essential for binding to the receptor, resulting in cell membrane fusion that allows viral entry into the host cell. The S protein monomer consists of S1 and S2 subunits or domains, with the receptor-binding domain (RBD) on the S1 subunit.

The receptor on the host cell is the ACE2 molecule, just as for SARS-CoV, and the RBD has to flip up like a hinge to allow the virus-receptor binding. Several antibodies have been identified that bind to the RBD of SARS-CoV or MERS-CoV, and these are being screened for neutralizing activity against the SARS-CoV-2 virus.

SARS-CoV-2 coronavirus, the virus which causes COVID-19, scientifically accurate 3D illustration showing surface spikes of the virus. Image Credit: Kateryna Kon / Shutterstock

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources

The discovery of neutralizing antibodies

The current study first teased out virus-specific memory B-cells from the convalescent serum of a COVID-19 patient and amplified the variable regions encoding the heavy and light chains. After producing recombinant IgG1 antibodies, they screened them for binding activity against SARS-CoV-2, with anti-SARS-CoV and another irrelevant antibody as the controls.

They found four different antibodies which bind the RBD of SARS-CoV-2 but not SARS-CoV, which indicates that they have distinct epitopes.

They then studied the strength of binding and binding affinity to the viral RBD and the neutralizing activity.

The results showed neutralizing activity for all four antibodies, with B38 being the most potent, followed by H4, H2, and B5 in that order. When competitive binding was performed, B38 and H4 actively competed with ACE2 for RBD binding, B5 showed partial competition, and H2 was a noncompetitive antibody.

The findings indicate that different monoclonal antibodies may have neutralizing activities through different mechanisms.

The blocking assay showed that two of the antibodies (B38 and H4) blocked RBD-ACE2 binding, presumably because they are attached to the interface between these two. They have different binding sites on the RBD, which overlap partially, allowing both to attach at the same time.

A mouse study explored the ability of the antibodies to help mice infected with the challenge, but which expressed ACE2 because of genetic engineering. These mice had a shorter course of illness when given a single dose of B38, but not H4 or a PBS control. The viral RNA load was lower, with both B38 and H4 administration relative to the PBS group.

Exploring the Mechanism of Action

A structural study was performed by crystallization and analysis of the B38-RBD complex. This shows that the RBD region responsible for antibody binding to the virus is poorly conserved between both SARS-CoV and SARS-CoV-2 viruses, which is why the B38 antibody is specific in binding to the latter virus.

They also superimposed the crystal structures of SARS-CoV-2 RBD-B38 and SARS-CoV, to find out why B38 inhibited viral binding to ACE2. They discovered that this antibody sterically hinders RBD-ACE2 binding. The RBD binding site has 18/21 amino acids that bind B38 and ACE2 exactly the same way, which is why this antibody blocks viral-receptor interactions completely.

While both SARS-CoV and SARS-CoV-2 use the ACE2 receptor for viral entry, the latter has a higher binding affinity to the receptor due to increased atomic interactions, which are due to different amino acids. These differences mean that neutralizing antibodies must be specific to the virus, and no study has reported an antibody that can cross-react with both by competing with the virus RBD for the ACE2 site. However, the CR3022 antibody found in convalescent SARS patient serum can bind RBD in the S protein of both viruses. It also neutralizes SARS-CoV.

A recent study shows, contrary to the findings of the current study that CR3022 does neutralize SARS-CoV-2 as well. The CR3022 binding site is independent of the ACE2 binding site. This discover could facilitate the development of cocktail antibodies that act via both RBD binding and ACE2 binding, inhibiting the viral entry into the host cell by different mechanisms in a synergistic manner.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources